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Uptake of a Fluorescent Dye as a Swift and Simple Indicator of Organelle Intactness : Import-competent Chloroplasts from Soil-grown Arabidopsis

Alexander Schulz, Jürgen Knoetzel, Henrik V. Scheller and Alexandra Mant

Plant Biochemistry Laboratory (JK,HVS,AM) and Plant Anatomy and Physiology Laboratory (AS), The Royal Veterinary and Agricultural University, Copenhagen, Denmark

Correspondence to: Alexandra Mant, Plant Biochemistry Laboratory, The Royal Veterinary & Agricultural University, 40, Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark. E-mail: ama{at}kvl.dk

We developed a rapid and reliable technique for specifically staining intact chloroplasts using the fluorescent dye carboxyfluorescein diacetate. Intact, import-competent chloroplasts were isolated simply and rapidly from soil-grown Arabidopsis thaliana plants, with yields of 20 ± 5 µg chlorophyll per g FW, greater than previously reported yields from soil-grown Arabidopsis. Traditional chloroplast isolation buffers sometimes contain low concentrations (<10 mM) sodium ascorbate as a general-purpose anti-oxidant, but we found that only Arabidopsis chloroplasts isolated in the presence of high concentrations (50–100 mM) of sodium ascorbate in the initial grinding buffer were import-competent. (J Histochem Cytochem 52:701–704, 2004)

Key Words: carboxyfluorescein diacetate • intact organelles • Arabidopsis • chloroplast • glucosinolates • protein import

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